Method for quantitatively detecting specific activity of MntC antigen in recombinant staphylococcus aureus vaccine or / and stock solution

Abstract

The invention discloses a method for quantitatively detecting the specific activity of a MntC antigen in a recombinant staphylococcus aureus vaccine or / and a stock solution. The method comprises the following steps: (1) coating an elisa plate by taking MntC-686 as a capture antibody; (2) adding a sample to be detected into the coated ELISA plate; (3) carrying out a chromogenic reaction by using HRP-MntC-A7, and measuring an OD450 / 630 nm value; and (4) preparing a standard curve by using the MntC reference, and calculating the specific activity of the MntC antigen in the detected sample according to the standard curve. According to the present invention, the fully human monoclonal antibody for specifically recognizing the recombinant staphylococcus aureus vaccine MntC protein is adopted as the coating antibody, and the anti-MntC protein fully human monoclonal antibody is adopted as the detection antibody; the kit can be used for detecting the specificity of the MntC antigen in a staphylococcus aureus vaccine or a stock solution, and has good stability and repeatability.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to a method for quantitatively detecting the specific activity of MntC antigen in a recombinant Staphylococcus aureus vaccine or a stock solution. Background Art

[0002] Staphylococcus aureus is the main pathogen causing hospital-acquired cross-infection, with high morbidity and mortality. It mainly causes osteomyelitis, endocarditis, septic arthritis, pneumonia, abscesses and toxic shock syndrome. Staphylococcus aureus can survive on dry surfaces, increasing the chance of infection. Infection with any type of Staphylococcus aureus can cause staphylococcal scalded skin syndrome, mainly because the skin tissue overreacts to toxins after bacterial endotoxins enter the human blood sample. Staphylococcus aureus can also cause bacteremia / sepsis, endangering human life. Among them, methicillin-resistant Staphylococcus aureus (MRSA) can secrete a variety of toxins, destroy normal humoral immunity and cellular immunity, reshape the host's immune environment, and develop multidrug resistance. At present, MRSA has become the Gram-positive bacteria with the highest infection rate in ICU wards, postoperative infections, burns, etc. It has become a difficult problem in clinical treatment, a serious threat to human health, and a major pathogen of hospital cross-infection. At present, infections caused by Staphylococcus aureus are usually treated with antibiotics such as penicillin, and vancomycin is used as an alternative therapy for penicillin-resistant strains. Vancomycin, as the drug of choice for the clinical treatment of Gram-positive bacterial infections, was once called the "last line" of clinical anti-infection. However, with the emergence of vancomycin-resistant Staphylococcus aureus in recent years, there is an urgent need to develop new anti-infection drugs. Therefore, the search for anti-MRSA drugs has become an important direction in the field of MRSA treatment research in recent years.

[0003] In the development of Staphylococcus aureus vaccine, antigen specific activity is an important indicator to measure the quality of antigen. It reflects the immune activity of the antigen per unit mass. High antigen specific activity means that the antigen has strong immunogenicity. In order to ensure the quality and stability of the recombinant Staphylococcus aureus vaccine and stock solution, it is necessary to test the antigen specific activity of the vaccine and stock solution.

[0004] The specific activity of the reference product is specified (1 ng is 1 YU). The double antibody sandwich enzyme-linked immunosorbent assay is used to draw a regression line with the reference product unit number (YU), and the test product unit number (YU) is calculated according to the standard curve equation, and then the specific activity of the test product is calculated according to the formula. Test product specific activity = test product (YU / mL) × dilution factor / test product protein concentration (mg / mL). If the specific activity value of the test product is less than 0.5×10 6YU / mg, the sample is judged to be unqualified. Summary of the invention

[0005] The object of the present invention is to provide a method for quantitatively detecting the MntC antigen specific activity in a recombinant Staphylococcus aureus vaccine or a stock solution, so as to solve the problem that there are few methods for detecting the antigen specific activity of Staphylococcus aureus vaccine in the prior art.

[0006] To achieve the above object, the present invention provides the following technical solutions: The present invention provides a method for quantitatively detecting the specific activity of MntC antigen in a recombinant Staphylococcus aureus vaccine or a stock solution, comprising the following steps: (1) Coating the ELISA plate with MntC-686 as the capture antibody; (2) Add the sample to be tested to the coated ELISA plate; (3) Use HRP-MntC-A7 for color development and measure OD 450 / 630 nm value; (4) Prepare a standard curve using the MntC reference product and calculate the specific activity of the MntC antigen in the test sample based on the standard curve.

[0007] Furthermore, the OD 450 / 630 nm The values ​​were measured as OD 450 nm and OD 630 nm value.

[0008] Furthermore, the specific activity of the MntC antigen is not less than 0.5×10 6 YU / mg is qualified.

[0009] Furthermore, after the ELISA plate in (1) was coated, it was refrigerated overnight, washed three times with 1×PBST, and blocked with 3% BSA for 1 h.

[0010] Furthermore, in (2), the plate needs to be washed three times with 1×PBST before the sample is added to the ELISA plate, and the plate needs to be incubated at 37° C. for 60 min after the sample is added to the ELISA plate.

[0011] Furthermore, HRP-MntC-A7 in (3) was added to the ELISA plate and incubated at 37°C for 60 min. After washing the plate three times with 1×PBST, TMB colorimetric solution was added for color development. After color development, 2 M sulfuric acid stop solution was added. TMB is tetramethylbenzidine. Furthermore, the standard curve in (4) is based on the concentration of the reference product as the horizontal axis and the OD as the horizontal axis. 450 / 630 nm The double logarithmic function constructed with the values ​​as the ordinate.

[0012] Furthermore, when the concentration of the standard curve reference substance in (4) is between 4.88 and 156.25 YU / mL, R 2 =0.9924.

[0013] Furthermore, the MntC-686 antibody includes a heavy chain and a light chain, and the heavy chain and light chain respectively contain three CDRs, wherein the heavy chain CDR1 GFTFDNYG, the heavy chain CDR2 IRSDGSNY, and the heavy chain CDR3 ARQAGDLLYLDL; the light chain CDR1QSVGTD, the light chain CDR2 STS, and the light chain CDR3 QQSYTTPLT.

[0014] Further, the MntC-686 antibody heavy chain and light chain also include FR regions, and the heavy chain and light chain respectively include four FR regions, wherein heavy chain FR1 EVQLVESGGGVVQPGRSLRLSCAAS, heavy chain FR2 MHWVRQAPGKGLEWVAI, heavy chain FR3 KHADSVKGRFSISRDNSRDTLFLQMTGLRVEDTAIYYC, heavy chain FR4 WGQGTLVTVSS; light chain FR1DIVMTQSPPSLSASVGDRITISCRTS, light chain FR2 LNWYQQKAGKAPELLVY, light chain FR3 SSLQSGVPSRFRGSGSGTEFTLTISSLQPEDFATYYC, light chain FR4 FGGGTKVDIK.

[0015] Further, the HRP-MntC-A7 antibody includes a heavy chain and a light chain, and the heavy chain and light chain respectively contain three CDRs, wherein the heavy chain CDR1 GFTFSSHG, the heavy chain CDR2 IWSDGTLK, the heavy chain CDR3 ARDPLTGYYIDH; the light chain CDR1NIGSKS, the light chain CDR2 DDS, the light chain CDR3 QVWDSSSDHWV.

[0016] Further, the HRP-MntC-A7 antibody heavy chain and light chain also include FR regions, and the heavy chain and light chain respectively contain four FR regions, wherein the heavy chain FR1 EVQLVESGGGVVQPGKSLRLSCAAS, heavy chain FR2 MHWVRQAPGKGLEWVAI, heavy chain FR3 YYSDSVKGRFTISRDDSKATVYLHMNSLRAEDTAVYYC heavy chain FR4 WGQGTLVTVSS; light chain FR1ELVLTQPPSVSVAPGKTARITCGGN, light chain FR2 VHWYQQKPGQAPVLVVY, light chain FR3 SDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYC, light chain FR4 FGGGTKVTVLGGG.

[0017] Further, the PBST is Na2 HPO 4 8 mM, NaCl 0.136 M, KH 2 PO 4 2mM, KCl 2.6mM, Tween-20 0.05%v / v.

[0018] Based on the above technical solution, the embodiments of the present invention can at least produce the following technical effects: The fully human monoclonal antibody that specifically recognizes the MntC protein of the recombinant Staphylococcus aureus vaccine is used as the coating antibody, and the fully human monoclonal antibody against the MntC protein is used as the detection antibody. The specific activity of the MntC antigen in the Staphylococcus aureus vaccine or stock solution can be detected, and it has good stability and repeatability. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying creative work.

[0020] Figure 1 4 is a linear verification result diagram of an embodiment of the present invention. DETAILED DESCRIPTION

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0023] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0024] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0025] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0026] The MntC-686 antibody includes a heavy chain and a light chain. The heavy chain and the light chain each contain three CDRs, including heavy chain CDR1GFTFDNYG, heavy chain CDR2 IRSDGSNY, heavy chain CDR3 ARQAGDLLYLDL; light chain CDR1 QSVGTD, light chain CDR2STS, light chain CDR3 QQSYTTPLT.

[0027] The heavy chain and light chain of the MntC-686 antibody also include FR regions, and the heavy chain and light chain respectively include four FR regions, wherein the heavy chain FR1 EVQLVESGGGVVQPGRSLRLSCAAS, heavy chain FR2 MHWVRQAPGKGLEWVAI, heavy chain FR3 KHADSVKGRFSISRDNSRDTLFLQMTGLRVEDTAIYYC, heavy chain FR4 WGQGTLVTVSS; light chain FR1DIVMTQSPPSLSASVGDRITISCRTS, light chain FR2 LNWYQQKAGKAPELLVY, light chain FR3 SSLQSGVPSRFRGSGSGTEFTLTISSLQPEDFATYYC, light chain FR4 FGGGTKVDIK.

[0028] The HRP-MntC-A7 antibody includes a heavy chain and a light chain. The heavy chain and the light chain each contain three CDRs, including heavy chain CDR1GFTFSSHG, heavy chain CDR2 IWSDGTLK, heavy chain CDR3 ARDPLTGYYIDH; light chain CDR1 NIGSKS, light chain CDR2DDS, light chain CDR3 QVWDSSSDHWV.

[0029] The heavy chain and light chain of the HRP-MntC-A7 antibody also include FR regions, and the heavy chain and light chain respectively include four FR regions, wherein the heavy chain FR1 EVQLVESGGGVVQPGKSLRLSCAAS, the heavy chain FR2 MHWVRQAPGKGLEWVAI, the heavy chain FR3 YYSDSVKGRFTISRDDSKATVYLHMNSLRAEDTAVYYC and the heavy chain FR4 WGQGTLVTVSS; the light chain FR1ELVLTQPPSVSVAPGKTARITCGGN, the light chain FR2 VHWYQQKPGQAPVLVVY, the light chain FR3 SDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYC and the light chain FR4 FGGGTKVTVLGGG.

[0030] PBST for Na 2 HPO 4 8 mM, NaCl 0.136 M, KH 2 PO 4 2mM, KCl 2.6mM, Tween-20 0.05%v / v.

[0031] Example 1 Optimization of detection conditions for capture and detection antibodies 1. Experimental methods The capture antibody MntC-686 was coated at different concentrations at 4°C overnight, washed three times with 1×PBST, and blocked with 3% BSA at 200 μL / well at 37°C for 1 hour. Washed three times with 1×PBST, incubated with MntC antigen at 37°C for 1 hour. Washed three times with 1×PBST, serially diluted the enzyme-labeled MntC-A7 monoclonal antibody, incubated at 37°C for 1 hour, washed, added TMB substrate colorimetric solution, developed at 37°C in the dark for 10 minutes, added 50 μL of 2 M sulfuric acid stop solution to each well, and detected OD with a microplate reader. 450 / 630nm value.

[0032] 2. Experimental results Criteria: (1) Calculate the OD ratio of each experimental well to the control well; (2) The OD ratio of the experimental well (P) to the control well (N) is greater than 2.1. The maximum P / N value is selected as the preferred combination.

[0033] As shown in Table 1, the capture antibody MntC-686 concentration is preferably 4 μg / mL, and the enzyme-labeled detection antibody MntC-A7 is diluted 1:1000 as a combination.

[0034] Table 1 P / N values ​​of capture antibody and detection antibody Example 2: Establishment of standard curve 1. Experimental methods The ELISA plate was coated with MntC-686 (4 μg / mL) as the capture antibody, 100 μL per well, coated overnight at 4°C, washed three times with 1×PBST, blocked with 3% BSA, 200 μL per well, blocked at 37°C for 1 h. The plate was washed three times with 1×PBST, and the MntC reference product was diluted 2-fold with 10 μg / mL as the starting concentration, 200 μL / well was added to the first column of the ELISA plate, 100 μL of the sample was taken from the first column and added to the second column until the last well was diluted, 100 μL per well for each dilution; the negative control was the diluent, 2 replicates, incubated at 37°C for 60 min.

[0035] After washing the plate three times with a plate washer, HRP-MntC-A7 was diluted with diluent at 1:1000, and 100 μL was added to each well of the ELISA plate, and incubated in a 37°C incubator for 60 min. The plate was washed three times with 1×PBST, protected from light, and 100 μL of TMB colorimetric solution was added to each well. The plate was developed at 37°C in a dark environment for 10 min, and 50 μL of 2 M sulfuric acid stop solution was added to each well. OD was detected using an ELISA reader. 450 / 630nm The standard curve was constructed with the concentration of the reference product as the horizontal axis and the OD value as the vertical axis, and the curve equation and correlation coefficient were obtained.

[0036] 2. Experimental results 2.1 Data processing Export detection OD 450 / 630nm Use EXCEL to calculate the OD of different concentrations of reference products 450 / 630nm The logarithm of the reference concentration is used as the horizontal axis, and the OD 450 / 630nm The logarithmic value constructs a double logarithmic function for the ordinate.

[0037] 2.2 Data Analysis When the reference concentration is 4.88-156.25 YU / mL, R 2 =0.9924, with good linearity, and the linearity is established.

[0038] Example 3: Methodological Validation 1. Exclusiveness verification (1) Experimental methods In order to evaluate the specific reaction of the established method, finished vaccine products, desorption reagents, and 1× PBST buffer were used as samples to verify the specificity of the method.

[0039] The finished vaccine, desorption reagent, and 1×PBST buffer were used as samples to be tested, respectively, and the concentration range of the reference product 4.88-156.25 YU / mL was used as the standard curve, and the double antibody sandwich ELISA experiment was performed according to the steps listed in Example 2. The data analysis was completed with reference to the analysis method shown in Example 2.

[0040] (2) Experimental results As shown in Table 2: OD of desorption reagent and 1× PBST buffer 450 / 630nm There is no difference, and the detection values ​​are all less than the lowest value of the standard curve, indicating that the desorption reagent has no effect on the detection results. The capture antibody MntC-686 is specific for MntC antigen recognition, and the method has good specificity.

[0041] Table 2 Specificity verification results 2. Intermediate precision verification (1) Experimental methods According to the calibration curve and operation steps established in Example 2, two persons took the same sample for testing at different times.

[0042] (2) Experimental results The relative standard deviation is within 10% (RSD≤10%), and the measured values ​​using the method of the present invention have high precision, as shown in Table 3.

[0043] Table 3 Intermediate precision verification results 4. Linear Verification (1) Experimental methods The experiment was repeated three times according to the calibration curve and operation steps established in Example 2, with the logarithm of the reference product concentration as the horizontal axis and the OD as the horizontal axis. 450 / 630nm Construct a double logarithmic function with the logarithmic value as the ordinate and calculate R 2 .

[0044] (2) Experimental results like Figure 1 As shown, the R 2 They are 0.9914, 0.9956, 0.9927, R 2 All of them are greater than 0.99, and the method has good linearity.

[0045] Example 5: Detection of MntC content in multiple batches of finished vaccine products and stock solutions (1) Experimental methods According to the standard curve and operating steps established in Example 2, three batches of vaccine products and three batches of MntC stock solutions were tested for specific activity.

[0046] (2) Experimental results If the specific activity of the sample is less than 0.5×10 6 YU / mg, the sample is judged to be unqualified. The MntC antigen specific activity test of the three batches of vaccine finished products was qualified, and the RSD was less than 10%, indicating that the process of different batches of finished products was stable, as shown in Table 4. The MntC antigen specific activity test of the three batches of stock solutions was qualified, and the RSD was less than 10%, indicating that the process of different batches of stock solutions was stable, as shown in Table 5.

[0047] Table 4 Results of MntC antigen specific activity test of finished vaccine products Table 5 Results of MntC stock solution antigen specific activity test Finally, it should be noted that: The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions described in the above embodiments may still be modified, or some or all of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for quantitatively detecting the specific activity of MntC antigen in a recombinant Staphylococcus aureus vaccine or a stock solution, characterized in that: The following steps are involved: (1) Coating the ELISA plate with MntC-686 as the capture antibody; (2) Add the sample to be tested to the coated ELISA plate; (3) Use HRP-MntC-A7 for color development and measure OD 450 / 630 nm value; (4) Prepare a standard curve using the MntC reference product and calculate the specific activity of the MntC antigen in the test sample based on the standard curve.

2. The method for quantitatively detecting the specific activity of MntC antigen in a recombinant Staphylococcus aureus vaccine or a stock solution according to claim 1, characterized in that: The specific activity of the MntC antigen is not less than 0.5×10 6 YU / mg is qualified.

3. The method for quantitatively detecting the specific activity of MntC antigen in a recombinant Staphylococcus aureus vaccine or a stock solution according to claim 1, characterized in that: After the ELISA plate was coated in (1), it was refrigerated overnight, washed three times with 1×PBST, and blocked with 3% BSA for 1 h.

4. The method for quantitatively detecting the specific activity of MntC antigen in a recombinant Staphylococcus aureus vaccine or a stock solution according to claim 1, characterized in that: In the above (2), the plate needs to be washed three times with 1×PBST before the sample is added to the ELISA plate, and the plate needs to be incubated at 37°C for 60 min after the sample is added to the ELISA plate.

5. The method for quantitatively detecting the specific activity of MntC antigen in a recombinant Staphylococcus aureus vaccine or a stock solution according to claim 1, characterized in that: The HRP-MntC-A7 in (3) was added to the ELISA plate and incubated at 37°C for 60 min. The plate was washed three times with 1×PBST and then TMB colorimetric solution was added for color development. After color development, 2 M sulfuric acid stop solution was added.

6. The method for quantitatively detecting the specific activity of MntC antigen in a recombinant Staphylococcus aureus vaccine or a stock solution according to claim 1, characterized in that: The standard curve in (4) is based on the concentration of the reference product as the horizontal axis and the OD as the horizontal axis. 450 / 630 nm The double logarithmic function constructed with the values ​​as the ordinate.

7. The method for quantitatively detecting the specific activity of MntC antigen in a recombinant Staphylococcus aureus vaccine or a stock solution according to claim 1, characterized in that: When the concentration of the standard curve reference material in (4) is between 4.88-156.25 YU / mL, R 2 =0.9924.

8. The method for quantitatively detecting the specific activity of MntC antigen in a recombinant Staphylococcus aureus vaccine or a stock solution according to claim 1, characterized in that: The MntC-686 antibody comprises a heavy chain and a light chain, wherein the heavy chain and the light chain respectively comprise three CDRs, wherein the heavy chain CDR1 is GFTFDNYG, the heavy chain CDR2 is IRSDGSNY, and the heavy chain CDR3 is ARQAGDLLYLDL; the light chain CDR1 is QSVGTD, the light chain CDR2 is STS, and the light chain CDR3 is QQSYTTPLT.

9. The method for quantitatively detecting the specific activity of MntC antigen in a recombinant Staphylococcus aureus vaccine or a stock solution according to claim 1, characterized in that: The HRP-MntC-A7 antibody comprises a heavy chain and a light chain, wherein the heavy chain and the light chain respectively comprise three CDRs, wherein the heavy chain CDR1 GFTFSSHG, the heavy chain CDR2 IWSDGTLK, the heavy chain CDR3 ARDPLTGYYIDH; the light chain CDR1 NIGSKS, the light chain CDR2 DDS, the light chain CDR3 QVWDSSSDHWV.

10. The method for quantitatively detecting the specific activity of MntC antigen in a recombinant Staphylococcus aureus vaccine or a stock solution according to claim 1, characterized in that: The PBST comprises Na2HPO4 8 mM, NaCl 0.136 M, KH2PO4 2 mM, KCl 2.6 mM, and Tween-20 0.05% v / v.

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